WO2019161294A1 - Ciblage du métabolisme des lipides et de l'oxydation des acides gras libres (ffa) pour traiter des maladies médiées par des lymphocytes t mémoires résidents (trm) - Google Patents

Ciblage du métabolisme des lipides et de l'oxydation des acides gras libres (ffa) pour traiter des maladies médiées par des lymphocytes t mémoires résidents (trm) Download PDF

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WO2019161294A1
WO2019161294A1 PCT/US2019/018341 US2019018341W WO2019161294A1 WO 2019161294 A1 WO2019161294 A1 WO 2019161294A1 US 2019018341 W US2019018341 W US 2019018341W WO 2019161294 A1 WO2019161294 A1 WO 2019161294A1
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disease
trm
acid
fabp4
cells
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PCT/US2019/018341
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Thomas S. Kupper
Rachael Clark
Youdong PAN
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Kupper Thomas S
Rachael Clark
Pan Youdong
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Priority to US16/970,879 priority Critical patent/US20200375965A1/en
Priority to EP19754942.1A priority patent/EP3755322A4/fr
Publication of WO2019161294A1 publication Critical patent/WO2019161294A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
    • A61K31/167—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide having the nitrogen of a carboxamide group directly attached to the aromatic ring, e.g. lidocaine, paracetamol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/17—Amides, e.g. hydroxamic acids having the group >N—C(O)—N< or >N—C(S)—N<, e.g. urea, thiourea, carmustine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A61K31/216—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acids having aromatic rings, e.g. benactizyne, clofibrate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/336—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having three-membered rings, e.g. oxirane, fumagillin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/4458—Non condensed piperidines, e.g. piperocaine only substituted in position 2, e.g. methylphenidate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis

Definitions

  • TRM tissue-resident memory T cells
  • CD8 + TRM provide a rapid antigen-specific immune response, creating an inflammatory and antiviral microenvironment that facilitates pathogen elimination 6 9 .
  • FFA exogenous lipid and free fatty acid
  • FFA Fatty Acid Binding Proteins
  • TRM Resident Memory T Cells
  • TRM -mediated diseases include many autoimmune and autoinflammatory diseases and disorders, which include diseases of skin (e.g., psoriasis, vitiligo, graft vs host disease, CTCL, contact dermatitis, alopecia areata, eczematous dermatitis), GI tract (e.g., Crohns disease, ulcerative colitis), lung (e.g., asthma), joint (e.g., rheumatoid arthritis, spondyloarthropathies), CNS (e.g., multiple sclerosis), and endocrine system (e.g., Type I diabetes), and more.
  • skin e.g., psoriasis, vitiligo, graft vs host disease, CTCL, contact dermatitis, alopecia areata, eczematous dermatitis), GI tract (e.g., Crohns disease, ulcerative colitis), lung (e.g., asthma
  • a TRM-mediated disease comprising administering a therapeutically effective amount of one or more inhibitors of exogenous lipid and free fatty acid uptake or of mitochondrial beta oxidation of internalized exogenous FFA (e.g., inhibitors of CD36 and/or FABP antagonists, e.g., inhibitors of FABP4 and/or FABP5) to a subject in need thereof.
  • a therapeutically effective amount of one or more inhibitors of exogenous lipid and free fatty acid uptake or of mitochondrial beta oxidation of internalized exogenous FFA e.g., inhibitors of CD36 and/or FABP antagonists, e.g., inhibitors of FABP4 and/or FABP5
  • the TRM-mediated diseases is selected from the gropu consisting of autoimmune and autoinflammatory diseases and disorders, e.g., diseases of skin (psoriasis, vitiligo, graft vs host disease, CTCL, contact dermatitis, alopecia areata, eczematous dermatitis), GI tract (Crohns disease, ulcerative colitis), lung (asthma), joint (rheumatoid arthritis, spondyloarthropathies), CNS (multiple sclerosis), and endocrine system (Type I diabetes).
  • diseases of skin psoriasis, vitiligo, graft vs host disease, CTCL, contact dermatitis, alopecia areata, eczematous dermatitis), GI tract (Crohns disease, ulcerative colitis), lung (asthma), joint (rheumatoid arthritis, spondyloarthropathies), CNS (
  • compositions comprising (i) one or more inhibitors of mitochondrial exogenous lipid or free fatty uptake and/or (ii) one or more inhibitors of mitochondrial fatty acid oxidation or metabolis, for use in treating or reducing risk of development or progression of an immune or inflammatory disease.
  • the disease is mediated by resident memory T cells (TRM).
  • TRM-mediated disease is an autoimmune or auto-inflammatory disease or disorder involving non-lymphoid tissue.
  • the disease is an autoimmune or auto-inflammatory disease or disorder is selected from one or more of the following: (a) diseases of the skin; (b) diseases of the gastrointestinal (GI) Tract; (c) endocrine or metabolic diseases (d) diseases of the lung; (e) diseases of the bones or joints or (f) diseases of the CNS.
  • the disease of the skin is selected from (a) psoriasis; (b) vitiligo; (c) graft vs host disease; (d) contact dermatitis; (e) alopecia areata; or (f) eczematous dermatitis.
  • the disease of the GI tract is Crohn's Disease, irritable bowel disease, or ulcerative colitis.
  • the disease of the lung is asthma.
  • the endocrine or metabolic disease is Type I diabetes (insulin dependent diabetes mellitus).
  • the disease of the bones or joints is rheumatoid arthritis or a spondyl arthropathy .
  • the disease of the CNS is multiple sclerosis.
  • the one or more inhibitors of mitochondrial exogenous lipid or free fatty uptake comprise an inhibitor or antagonist of CD36 or a Fatty Acid Binding Protein (FABP).
  • FBP Fatty Acid Binding Protein
  • the inhibitor or antagonist of FABP is an inhibitor or antagonist of FABP4 or FABP5.
  • the inhibitor or antagonist of FABP4 or FABP5 is carbazole butanoic acid, aryl sulfonamide, sulfonylthiophene derivative, 4-hydroxypyrimidine, tetrahydrocarb azole derivative, 2,3-dimethylindole derivative, benzoylbenzene, biphenyl - alkanoic acid derivative, 2-oxazole-alkanoic acid derivative, tetrahydropyrimidone, pyridone, pyrazinone, aryl carboxylic acid, tetrazole, triazolopyrimidinone, BMS309403; pyrazole, 4- ⁇ [2-(methoxycarbonyl)-5-(2-thienyl)-3-thienyl]amino ⁇ -4-oxo-2-butenoic acid or ((2 '-(5 -e
  • the one or more inhibitors of mitochondrial fatty acid beta oxidation or metabolism is an inhibitor of carnitine palmitoyltransferase 1 (CPT1).
  • CPT1 carnitine palmitoyltransferase 1
  • the inhibitor of CPT1 is etomoxir; 2-tetradecylglycidic acid (TDGA); ST1326 (Teglicar); or perhexiline (2-(2,2-dicyclohexylethyl)piperidine) perhexiline (2-(2,2- dicyclohexylethyl)piperidine), or a derivative thereof, or an inhibitory nucleic acid.
  • the methods include administering a PPAR gamma inhibitor.
  • the PPAR gamma inhibitor is GW9662, or a derivative thereof.
  • the disease is a disease of the skin, and administration of the one or more inhibitors is by topical delivery.
  • the composition is formulated for topical administration.
  • FIGS 1A-G Skin CD8 + tissue-resident memory T cells (TRM) display increased expression of fatty acid binding protein 4 (FABP4) and FABP5.
  • A Principal component analysis (PCA) of gene-expression data for CD8 + T cell subtypes. Each timepoint represents an individual experiment wherein mRNA was pooled from 15-20 mice from 3-4 independent biological groups (5 mice/group).
  • B Pearson correlation coefficients among CD8 + T cell subtypes.
  • C Heatmap of differentially expressed genes selected from a pair-wise comparison between OT-I TRM (day 30) and TCM. Genes are shown with color indicating z-score across rows.
  • D Quantitative real-time PCR (qRT-PCR) analysis of Fabp4 and Fabp5 expression in TN, TCM, TEM and TRM (day 30).
  • E Quantitative real-time PCR analysis of Fabp4 and Fabp5 gene expression in skin CD103 and CDl03 + TRM (day 30).
  • F qRT-PCR analysis of I’pary expression in TN, TCM, TEM and TRM (day 30).
  • G Effect of lentiviral l’pcirr siRNA knockdown on Fabp4 and Fabp5 expression in OT-I CD8 + TRM. Graphs in D-G show mean ⁇ s. d.
  • T cells from 15-20 mice were pooled for each group ns, not significant, **p ⁇ 0.01.
  • FIGS 2A-H Loss of Fabp4 and Fabp5 decreases fatty acid uptake and metabolism by CD8 + TRM and impairs their long-term maintenance.
  • A Representative histograms and average MFI of Bodipy FL C16 uptake by TN, TCM, TEM or TRM (day 30). Graphs show mean ⁇ s. d. of 5 mice.
  • B Representative histograms and average MFI of Bodipy FL C16 uptake in OT-I WT and Fabp4/5 dKO TRM with or without pre-incubation with unlabelled palmitate. Graphs show mean ⁇ s. d. of 5 mice.
  • F Oxygen-consumption rate (OCR) of OT-I TCM, TRM and Fabp4/5 dKO TRM (day 30) under basal conditions and in response to indicated mitochondria inhibitors. Results were normalized to those of control cells in the presence of BSA. Graphs show mean ⁇ s. d. of triplicates.
  • G Effect of lentiviral Cptla siRNA knockdown on OT-I CD8 + TRM survival in vivo in infected tissue.
  • H Representative dot plots and enumeration of WT and Fabp4/5 dKO TCM and TRM 45 days post infection. Cells were gated on VACV- specific pentamer + CD8 + T cells. Symbols represent individual mice in E, G, H. ns, not significant, *p ⁇ 0.05, **p ⁇ 0.01
  • FIGS 3A-G Skin CD8 + TRM lacking Fabp4 and Fabp5 fail to protect mice against viral infectious challenge.
  • A Schematic of experimental design.
  • C-D Representative dot plots and quantification of IFN-g secretion by OT-I WT or Fabp4/5 dKO TRM. Symbols represent individual mice.
  • E Schematic of experimental design.
  • F-G Body weight (BW) (F) and survival measurements (G) of WR- VACV challenged mice, with or without FTY720 treatment.
  • FIG. 4A-D Human skin CD8 + TRM demonstrate increased expression of FABP4 and FABP5.
  • A-B Representative histograms and average MFI of intracellular staining of FABP4 (A) and FABP5 (B) in human PBMC TN, TCM, and TEM compared to facial skin TRM. Graphs show mean ⁇ s. d. of 5 human individuals.
  • C Hematoxylin and eosin (H&E)-staining of a human psoriatic lesion. Scale bar, 100 pm.
  • FIGS. 5A-F CD8 + TRM display increased gene expression of Fabp4 and Fabp5.
  • OT-I Thyl. l + cells were intravenously transferred into Thyl.2 + recipient mice 1 day before mice were infected with 2 x 10 6 p.f.u. VAC V OVA by intra-tracheal infection.
  • B Venn diagram analysis of genes differentially expressed in pairwise comparisons between OT-I TCM, TEM and TRM (day 30) relative to that of TN (fold change cutoff, >2).
  • C Absolute qRT-PCR analysis of Fabp gene expression in TRM (day 30). ND, not detectable.
  • D qRT-PCR assessments oiFabp4 and Fabp5 levels in indicated T cell subsets.
  • OT-I Thyl.l + cells were intravenously transferred into Thyl.2 + recipient mice 1 day before mice were infected with 2 x 10 6 p.f.u. VAC V OVA by intra-tracheal infection.
  • mice were sacrificed.
  • TCM and TEM were sorted from spleen and TRM were sorted from lung.
  • E qRT-PCR analysis of Ppar knockdown efficiency by lentiviral vector encoding two specific and one scrambled siRNA in OT-I CD8 + TRM.
  • F qRT-PCR analysis of Fabp4 and Fabp5 gene expression in OT-I CD8 + TRM from mice treated with or without GW9662.
  • Graphs in C, D, E, F show mean ⁇ s. d. from triplicates b-actin was used as internal control and mRNA was normalized to TN samples (D) or TRM with scrambled siRNA transduction (E) or without GW9662 treatment (F).
  • T cells from 15-20 mice were pooled for each sample ns, not significant, **p ⁇ 0.01.
  • FIGS 6A-D Relative contribution of FABP4 and FABP5 in fatty acid acquisition and long-term maintenance of skin CD8 + TRM.
  • A Average MFI of Bodipy FL C16 uptake by OT-I WT, Fabp4 +l Fabp5 +l Fabp4 j and FabpS 1 TRM. Graphs show mean ⁇ s. d. of 5 mice per group.
  • B-D Enumeration of OT-I WT, Fabp4 +l Fabp5 +l Fabp4 j and FabpS 1 TRM at different timepoints post VACVOVA infection.
  • Thyl.2 + CD45.2 + recipient mice were given a 1 :1 ratio of naive OT-I Thyl. l + CD45. l + WT and OT-I Thyl. l + CD45.2 + Fabp4/5 knockout Fabp4 +l Fabp5 +l , Fabp4 ! or babpS 1 ) cells one day before infection with 2 x 10 6 p.f.u. VACVOVA by skin scarification. Relative percentages of the two T cell populations were analyzed longitudinally by flow cytometry. Graphs show mean ⁇ s. d. of 5 mice per group ns, not significant.
  • FIGS 7A-C OT-I Fabp4/5 dKO effector T cells (T eff ) display similar proliferative capacity and tissue-homing receptor expression to WT counterparts.
  • A Quantification of Ki67 + cells in OT-I TN, T eff , TCM, TEM and TRM. Graphs show mean ⁇ s. d. of 5 mice per group.
  • B Average MFI of Bodipy FL C16 uptake by OT-I TN, T eff , TCM, TEM or TRM. Graphs show mean ⁇ s. d. of 5 mice per group. *p ⁇ 0.05, **p ⁇ 0.01.
  • Thyl.2 + CD45.2 + recipient mice were given a 1 :1 ratio of CFSE-labeled naive OT-I Thyl.l + CD45.l + WT and OT-I Thyl.l + CD45.2 + Fabp4/5 dKO cells one day before mice were infected with 2 x 10 6 p.f.u. VACVOVA by skin scarification.
  • OT-I Fabp4/5 dKO TRM show a similar surface protein expression phenotype to WT counterparts.
  • FIGS 9A-B Effect of Ppary lentiviral siRNA knockdown or PPARy inhibition on CD8 + TRM maintenance in peripheral tissue.
  • A Enumeration of OT-I CD8 + TRM transduced with scrambled siRNA or siRNA targeting Ppary.
  • Ppary siRNA transduced OT-I cells (together with the same number of congenically scrambled siRNA transduced OT-I cells) were cotransferred into recipient mice that were previously infected with VACVOVA by skin scarification. At 40 days later, mice were sacrificed and the number of siRNA transduced OT - I cells in the infected skin tissue were harvested and enumerated by flow cytometry based on the GFP marker.
  • mice Enumeration of OT-I CD8 + TRM in infected skin from mice treated with or without GW9662.
  • Thyl.2 + CD45.2 + recipient mice were infected with 2 x 10 6 p.f.u. VACVOVA by skin scarification. 40 days later, mice were treated with GW9662 daily by intradermal injection for 5 days, after which mice were sacrificed and TRM were enumerated and analyzed by flow cytometry.
  • Graphs show mean ⁇ s. d. of 5 mice per group. **p ⁇ 0.01.
  • FIGS 10A-B Gene expression profile of OT-I Fabp4/5 dKO TRM.
  • A Principal component analysis (PC A) of gene-expression data for skin infiltrating T cells isolated at day 10 and day 30 post infection.
  • B Differentially expressed genes selected from a pairwise comparison between OT-I Fabp4/5 dKO TRM and OT-I WT TRM.
  • Thyl.2 + CD45.2 + recipient mice were given a 1 :l ratio of naive OT-I Thyl.l + CD45. l + WT and OT-I Thyl. l + CD45.2 + Fabp4/5 dKO cells one day before mice were infected with 2 x 10 6 p.f.u.
  • OT-I CD8 + TRM were sorted from infected skin sites for gene microarray.
  • C qRT-PCR analysis of genes involved in anti-inflammatory responses ( 1110 and Socs2 ), cell apoptosis ( Caspase3 and Bcl2113 ) as well as immune responses ( Ccr8 , Cc/27, II IrP 116 , Cxcll3, Cxcll2 , Cell , Gzmc , Gzma) in OT-I Fabp4/5 dKO TRM compared to WT TRM.
  • Graphs show mean ⁇ s. d. from triplicates b-actin was used as internal control and mRNA was normalized to OT-I WT TRM.
  • mRNA was pooled from 15 mice from 3 independent biological groups (5 mice/group). **p ⁇ 0.01.
  • FIGS 11A-D Effect of Cptla lentiviral siRNA knockdown or CPT1A inhibition on OT-I CD8 + TRM maintenance in peripheral tissue.
  • A qRT-PCR analysis of Cptla lentiviral siRNA knockdown efficiency in OT-I CD8 + TRM.
  • B-C Enumeration of OT-I WT and OT-I Fabp4/5 dKO TRM in infected skin from mice treated with or without etomoxir (B) and trimetazidine (C).
  • Thyl.2 + CD45.2 + recipient mice were given a 1 : 1 ratio of naive OT-I Thyl. l + CD45.l + WT and OT-I Thyl.
  • mice were administered etomoxir or trimetazidine daily by intradermal injection for 5 days from day 40 post infection, after which mice were sacrificed and TRM were analyzed by flow cyto etry and enumerated. Symbols represent individual mice.
  • D Oxygen-consumption rate (OCR) and extracellular acidification rate (ECAR) as measured by the Seahorse assay of skin infiltrating T cells sorted at indicated timepoints post infection under basal conditions.
  • Graphs show mean ⁇ s. d. of triplicates ns, not significant, **p ⁇ 0.01.
  • FIGS 12A-C Skin CD8 + TRM residing in distal infection sites are deficient in long-time survival and virus clearance.
  • B Enumeration of OT-I WT and OT-I Fabp4/5 dKO cells infiltrating distal infection sites at different timepoints post VACVOVA infection.
  • Thyl.2 + CD45.2 + recipient mice were given a 1 :l ratio of naive OT-I Thyl.l + CD45. l + WT and OT-I Thyl.
  • mice 25 days later, mice were rechallenged with VACVOVA at left ear by skin scarification.
  • FIGS 13A-B Fatty acid uptake and T cell survival of cd36-/- TRM cells.
  • A Average MFI of Bodipy FL C16 uptake in OT-I WT and cd36-/- TRM with or without pre-incubation with unlabelled palmitate. Graphs show mean ⁇ s. d. of 5 mice.
  • B Enumeration of OT-I WT and cd36-/- TCM and TRM at different timepoints post infection. Symbols represent individual mice ns, not significant, **p ⁇ 0.01.
  • Figures 14A-B Fatty acid uptake and T cell survival of Fabp4-/-/Fabp5-/- cd36-/- TRM cells.
  • A Average MFI of Bodipy FL C16 uptake in OT-I WT, Fabp4-/-/Fabp5-/-, cd36-/- and Fabp4-/-/Fabp5-/- cd36-/- TRM with or without pre-incubation with unlabelled palmitate.
  • Graphs show mean ⁇ s. d. of 5 mice.
  • TRM depend on exogenous FFA for survival and function, and do not persist in tissues if they cannot utilize this energy source. Effect of FAO inhibition on contact hypersensitivity (CHS) reaction post DNFB re-exposure.
  • DNFB 20 ul, 0.25% in acetone:olive oil (aOO)(3:lv/v); Etomoxir (1 ug / site); Trimetazidine (10 ug/site).
  • N l0 mice per group.
  • TRM Tissue-resident memory T cells persist indefinitely in epithelial barrier tissues and protect the host against pathogens 1 4 .
  • the biological pathways that enable the long- term survival of TRM are obscure 4,5 .
  • CD8+ TRM generated by viral infection of skin differentially express high levels of several molecules mediating lipid uptake and intracellular transport, including fatty acid binding proteins 4 and 5 (FABP4 and FABP5).
  • FFA free fatty acid
  • CD8+ TRM but not CD8+ TCM demonstrated increased mitochondrial oxidative metabolism in the presence of exogenous FFA; this increase was not seen in Fabp4/5 dKO CD8+ TRM.
  • Persistence of CD8+ TRM in skin was strongly diminished by inhibition of mitochondrial FFA b-oxidation in vivo.
  • skin CD8+ TRM lacking Fabp4/5 were less effective at protecting mice from cutaneous viral infection
  • lung Fabp4/5 dKO CD8+ TRM generated by skin VACV infection were less effective at protecting mice from a lethal pulmonary challenge with VACV.
  • TRM pathogenic TRM mediate tissue-specific immune-mediated diseases as diverse as psoriasis and vitiligo, asthma, inflammatory bowel disease, rheumatoid and spondylo-arthritis, and insulin-dependent diabetes (see, e.g., Park and Kupper, Nat Med. 2015 Jul;2l(7):688-97). It was hypothesized that the difficulty in achieving durable remission in these diseases is because the genetic program of TRMs is focused on maintaining their indefinite survival in tissues. While TRMs’ disease-causing activity can be transiently blocked with immune suppressive drugs, there is currently no means of dislodging these pathogenic cells from tissue. As a result, these diseases are chronic and relapsing.
  • CD8+ TRM in skin depended upon uptake of exogenous free fatty acids (FFA), which they used for mitochondrial b oxidation and ATP generation. If either free fatty acid uptake or mitochondrial b oxidation were blocked, CD8+ TRM did not survive in peripheral tissue. Further, as shown herein, blocking TRM lipid uptake and metabolism can be used to dislodge pathogenic TRM from tissue, providing a durable treatment for TRM- mediate immune and inflammatory diseases.
  • FFA free fatty acids
  • the present disclosure provides methods for treating subjects with TRM mediate tissue-specific immune-mediated diseases including (a) diseases of the skin; (b) diseases of the gastrointestinal (GI) Tract; (c) endocrine or metabolic diseases (d) diseases of the lung; (e) diseases of the bones or joints or (f) diseases of the CNS (see, e.g., Park and Kupper, Nat Med. 2015 Jul;2l(7):688-97).
  • Diseases of the can include (a) psoriasis; (b) vitiligo; (c) graft vs host disease; (d) contact dermatitis; (e) alopecia areata; or (f) eczematous dermatitis.
  • Diseases of the GI tract can include Crohn's Disease, irritable bowel disease, or ulcerative colitis.
  • Diseases of the lung can include asthma.
  • Endocrine or metabolic diseases can include Type I diabetes (insulin dependent diabetes mellitus).
  • Disease of the bones or joints can include rheumatoid arthritis or a spondylarthropathy.
  • Diseases of the CNS can include multiple sclerosis (MS).
  • to“treat” means to ameliorate at least one symptom of the disorder.
  • the methods include identifying a subject who has a TRM mediate tissue-specific immune-mediated disease as described herein. Such subjects can be identified by one of skill in the art using known diagnostic methodology.
  • a subject treated by a method described herein does not have insulin- dependent diabetes exclude diabetes, psoriasis, or MS.
  • CD36 inhibitors including sulfo-N-succinimidyl oleate (SSO); Ursolic acid; AP5055 or AP5258 (Arteria, Geloen et ak, PLoS ONE 7(5): e37633); alvianolic acid B (SAB) SAB or its metabolites, such as rosmarinic acid (RA) and sodium danshensu (DSS); 3-cinnamoyl indole, and 13 pentyl berberine (Xu, Y et al, Anal Biochem 400(2): 207-212 (2010)): S-cirinamoy Indole 13- enty; hexarelin (Demers A et al, Biochem J.
  • SSO sulfo-N-succinimidyl oleate
  • Ursolic acid AP5055 or AP5258
  • SAB alvianolic acid B
  • RA rosmarinic acid
  • DSS sodium danshens
  • the FABP inhibitor can be a FABP4 and/or a FABP5 inhibitor. In some embodiments, the FABP inhibitor is a FABP4 inhibitor.
  • the FABP4 inhibitor can be, e.g., a carbazole butanoic acid, aryl sulfonamide, sulfonyl thiophene derivative, 4-hydroxypyrimidine, tetrahydrocarb azole derivative, 2,3-dimethylindole derivative, benzoylbenzene, biphenyl - alkanoic acid derivative, 2-oxazole-alkanoic acid derivative, tetrahydropyrimidone, pyridone, pyrazinone, aryl carboxylic acid, tetrazole, triazolopyrimidinone, and indole derivative.
  • a carbazole butanoic acid e.g., a carbazole butanoic acid, aryl sulfonamide, sulfonyl thiophene derivative, 4-hydroxypyrimidine, tetrahydrocarb azole derivative, 2,3-dimethylindole derivative, benzo
  • the FABP4 inhibitor is (BMS309403, Bristol Myers Squibb, described in Sulsky et al., Bioorganic & Medicinal Chemistry Letters 17(12), 3511-3515 (2007)); pyrazole, 4- ⁇ [2- (methoxycarbonyl)-5-(2-thienyl)-3-thienyl]amino ⁇ -4-oxo-2-butenoic acid or ((2'-(5-ethyl-3,4- diphenyl-lH-pyrazol-l-yl)(l,l '-biphenyl)-3-yl)oxy)-acetic acid.
  • the FABP inhibitor is a FABP5 inhibitor.
  • the FABP5 inhibitor can be, e.g., an indole derivative (Lehmann et al 2004), triazolopyrimidinone derivative (Schering Corporation, PCT/US2009/063787), or Pyrazole.
  • Other FABP5 inhibitors include SBFI26 (alpha-2,4- diphenyl cy cl obutane-l, 3 -dicarboxylic acid mono- 1 -naphthyl ester) and other a-truxillic acid derivatives, e.g., SBFI50 (alpha-2, 4-diphenylcyclobutane-l,3-dicarboxylic acid mono-2- naphthyl ester), SBFI60 (alpha-2, 4-diphenylcyclobutane-l,3-dicarboxylic acid mono-l- naphthyl amide), and SBFI62 (2,4-diphenylcyclobutane-l,3-dicarboxylic acid di-l-
  • the FABP4 inhibitor can be an inhibitory nucleic acid, e.g., as described herein, e.g., a small interference RNA (siRNA), in particular, a small hairpin RNA (shRNA).
  • shRNA against FABP4 comprises a nucleic acid sequence of (5 '-AUACUGAGAUUUCCUUC AU-3 ') SEQ ID NO: 1 (Haijes et al., Oncogene. 2017 Feb 16; 36(7): 912-921).
  • CPT1 inhibitors and methods for administering the same are known in the art; see, e.g., US2016/0102058.
  • CPT1 inhibitors include 2-(6-(4-chlorophenoxy)-hexyl)-oxirane-2 -carboxylic acid ethyl ester (etomoxir) and its analog 2-tetradecylglycidic acid (TDGA) (Morillas et al., Biochem. J. (2000) 351, 495-502); aminocarnitine and ST 1326 (Teglicar), a long-chain carbamoyl aminocamitine derivative (Giannessi et al., J Med Chem 2003; 46: 303-309); and perhexiline (2-(2,2-dicyclohexylethyl)piperidine) (Horgan et al., U.S. Pat. No.
  • a number of derivatives of perhexiline are known in the art, including N-Substituted derivatives (see, e.g., WO 2007/096251); derivatives in which the piperidine has been replaced by other amine-bearing groups (see, e.g., LeClerc et al., J. Med. Chem., Vol. 25, pp. 709-714, 1982); derivatives in which the carbon separating the two cyclohexyl groups has been substituted with a hydroxyl group (see, e.g., Tilford and van Campen, J. Am. Chem. Soc., Vol.
  • hydroxyperhexiline i.e., 4-[l-(cyclohexyl)-2-(2-piperidinyl)ethyl]cyclohexanol; CAS Registry No 89787-89-3
  • trans-hydroxyperhexiline i.e., trans-4-[l-(cyclohexyl)-2-(2- piperidinyl)ethyl]cyclohexanol; CAS Registry No 917877-74-8
  • cis-hydroxyperhexiline i.e., cis-4-[l-(cyclohexyl)-2-(2-piperidinyl)ethyl]cyclohexanol; CAS Registry No 917877- 73-7).
  • inhibitors of mitochondrial beta-oxidation of fatty acids include methyl palmoxirate, metoprolol, hydrazonopropionic acid, 4-bromocrotonic acid, ranolazine, hypoglycin, dichloroacetate, methylene cyclopropyl acetic acid, or beta-hydroxybutyrate (LTS 7,510,710).
  • Other examples of inhibitors of fatty acid beta-oxidation include pirprofen (Geneve et ah, J Pharmacol Exp Ther. l987;242:l 133—1137) and amiodarone (Fromenty et ah, J Pharmacol Exp Ther.” 1990;255:1371-6).
  • Inhibitory Nucleic Acids useful in the present methods and compositions include antisense oligonucleotides, ribozymes, siRNA compounds, single- or double-stranded RNA interference (RNAi) compounds such as siRNA compounds, modified bases/locked nucleic acids (LNAs), peptide nucleic acids (PNAs), and other oligomeric compounds or oligonucleotide mimetics that hybridize to at least a portion of the target nucleic acid and modulate its function.
  • RNAi RNA interference
  • the inhibitory nucleic acids include antisense RNA, antisense DNA, chimeric antisense oligonucleotides, antisense oligonucleotides comprising modified linkages, interference RNA (RNAi), short interfering RNA (siRNA); a micro, interfering RNA (miRNA); a small, temporal RNA (stRNA); or a short, hairpin RNA (shRNA); small RNA- induced gene activation (RNAa); small activating RNAs (saRNAs), or combinations thereof.
  • RNAi interference RNA
  • siRNA short interfering RNA
  • miRNA micro, interfering RNA
  • shRNA small, temporal RNA
  • shRNA small RNA- induced gene activation
  • RNAa small activating RNAs
  • Exemplary sequences for the target nucleic acids include the following.
  • the inhibitory nucleic acids are 10 to 50, 10 to 20, 10 to 25, 13 to 50, or 13 to 30 nucleotides in length.
  • One having ordinary skill in the art will appreciate that this embodies inhibitory nucleic acids having complementary portions of 10, 11, 12, 13, 14, 15, 16,
  • the inhibitory nucleic acids are 15 nucleotides in length. In some embodiments, the inhibitory nucleic acids are 12 or 13 to 20, 25, or 30 nucleotides in length.
  • the inhibitory nucleic acids are 12 or 13 to 20, 25, or 30 nucleotides in length.
  • the inhibitory nucleic acids useful in the present methods are sufficiently complementary to the target RNA, i.e., hybridize sufficiently well and with sufficient specificity, to give the desired effect.
  • “Complementary” refers to the capacity for pairing, through hydrogen bonding, between two sequences comprising naturally or non-naturally occurring bases or analogs thereof. For example, if a base at one position of an inhibitory nucleic acid is capable of hydrogen bonding with a base at the corresponding position of a RNA, then the bases are considered to be complementary to each other at that position. 100% complementarity is not required.
  • Routine methods can be used to design an inhibitory nucleic acid that binds to the target sequence with sufficient specificity.
  • the methods include using bioinformatics methods known in the art to identify regions of secondary structure, e.g., one, two, or more stem-loop structures, or pseudoknots, and selecting those regions to target with an inhibitory nucleic acid.
  • bioinformatics methods known in the art to identify regions of secondary structure, e.g., one, two, or more stem-loop structures, or pseudoknots, and selecting those regions to target with an inhibitory nucleic acid.
  • “gene walk” methods can be used to optimize the inhibitory activity of the nucleic acid; for example, a series of oligonucleotides of 10-30 nucleotides spanning the length of a target RNA can be prepared, followed by testing for activity.
  • gaps e.g., of 5-10 nucleotides or more, can be left between the target sequences to reduce the number of oligonucleotides synthesized and tested.
  • GC content is preferably between about 30-60%. Contiguous runs of three or more Gs or Cs should be avoided where possible (for example, it may not be possible with very short (e.g., about 9-10 nt) oligonucleotides).
  • the inhibitory nucleic acid molecules can be designed to target a specific region of the RNA sequence.
  • a specific functional region can be targeted, e.g., a region comprising a known RNA localization motif (i.e., a region complementary to the target nucleic acid on which the RNA acts).
  • highly conserved regions can be targeted, e.g., regions identified by aligning sequences from disparate species such as primate (e.g., human) and rodent (e.g., mouse) and looking for regions with high degrees of identity. Percent identity can be determined routinely using basic local alignment search tools (BLAST programs) (Altschul et ak, J. Mol.
  • inhibitory nucleic acid compounds are chosen that are sufficiently complementary to the target, i.e., that hybridize sufficiently well and with sufficient specificity (i.e., do not substantially bind to other non-target RNAs), to give the desired effect.
  • hybridization means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases.
  • adenine and thymine are complementary nucleobases which pair through the formation of hydrogen bonds.
  • Complementary refers to the capacity for precise pairing between two nucleotides. For example, if a nucleotide at a certain position of an oligonucleotide is capable of hydrogen bonding with a nucleotide at the same position of a RNA molecule, then the inhibitory nucleic acid and the RNA are considered to be complementary to each other at that position.
  • the inhibitory nucleic acids and the RNA are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotides which can hydrogen bond with each other.
  • “specifically hybridisable” and“complementary” are terms which are used to indicate a sufficient degree of complementarity or precise pairing such that stable and specific binding occurs between the inhibitory nucleic acid and the RNA target. For example, if a base at one position of an inhibitory nucleic acid is capable of hydrogen bonding with a base at the corresponding position of a RNA, then the bases are considered to be complementary to each other at that position. 100% complementarity is not required.
  • a complementary nucleic acid sequence need not be 100% complementary to that of its target nucleic acid to be specifically hybridisable.
  • a complementary nucleic acid sequence for purposes of the present methods is specifically hybridisable when binding of the sequence to the target RNA molecule interferes with the normal function of the target RNA to cause a loss of activity, and there is a sufficient degree of complementarity to avoid non-specific binding of the sequence to non-target RNA sequences under conditions in which specific binding is desired, e.g., under physiological conditions in the case of in vivo assays or therapeutic treatment, and in the case of in vitro assays, under conditions in which the assays are performed under suitable conditions of stringency.
  • stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and more preferably less than about 250 mM NaCl and 25 mM trisodium citrate.
  • Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, and more preferably at least about 50% formamide.
  • Stringent temperature conditions will ordinarily include temperatures of at least about 30° C, more preferably of at least about 37° C, and most preferably of at least about 42° C.
  • Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art.
  • concentration of detergent e.g., sodium dodecyl sulfate (SDS)
  • SDS sodium dodecyl sulfate
  • Various levels of stringency are accomplished by combining these various conditions as needed.
  • hybridization will occur at 30° C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS.
  • hybridization will occur at 37° C in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 pg/ml denatured salmon sperm DNA (ssDNA).
  • hybridization will occur at 42° C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 pg/ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art.
  • wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature.
  • stringent salt concentration for the wash steps will preferably be less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate.
  • Stringent temperature conditions for the wash steps will ordinarily include a temperature of at least about 25° C, more preferably of at least about 42° C, and even more preferably of at least about 68° C.
  • wash steps will occur at 25° C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 42° C. in 15 mM NaCl, 1.5 mM tri sodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 68° C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations on these conditions will be readily apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al.
  • the inhibitory nucleic acids useful in the methods described herein have at least 80% sequence complementarity to a target region within the target nucleic acid, e.g., 90%, 95%, or 100% sequence complementarity to the target region within an RNA.
  • a target region within the target nucleic acid e.g. 90%, 95%, or 100% sequence complementarity to the target region within an RNA.
  • an antisense compound in which 18 of 20 nucleobases of the antisense oligonucleotide are complementary, and would therefore specifically hybridize, to a target region would represent 90 percent complementarity.
  • Percent complementarity of an inhibitory nucleic acid with a region of a target nucleic acid can be determined routinely using basic local alignment search tools (BLAST programs) (Altschul et al., J. Mol.
  • Inhibitory nucleic acids that hybridize to an RNA can be identified through routine experimentation. In general the inhibitory nucleic acids must retain specificity for their target, i.e., must not directly bind to, or directly significantly affect expression levels of, transcripts other than the intended target.
  • inhibitory nucleic acids please see US2010/0317718 (antisense oligos); US2010/0249052 (double-stranded ribonucleic acid (dsRNA)); US2009/0181914 and US2010/0234451 (LNAs); US2007/0191294 (siRNA analogues); US2008/0249039 (modified siRNA); and WO2010/129746 and W02010/040112 (inhibitory nucleic acids).
  • the inhibitory nucleic acids are antisense oligonucleotides.
  • Antisense oligonucleotides are typically designed to block expression of a DNA or RNA target by binding to the target and halting expression at the level of transcription, translation, or splicing.
  • Antisense oligonucleotides of the present invention are complementary nucleic acid sequences designed to hybridize under stringent conditions to an RNA. Thus, oligonucleotides are chosen that are sufficiently complementary to the target, i.e., that hybridize sufficiently well and with sufficient specificity, to give the desired effect.
  • the nucleic acid sequence that is complementary to a target RNA can be an interfering RNA, including but not limited to a small interfering RNA (“siRNA”) or a small hairpin RNA (“shRNA”).
  • interfering RNA including but not limited to a small interfering RNA (“siRNA”) or a small hairpin RNA (“shRNA”).
  • siRNA small interfering RNA
  • shRNA small hairpin RNA
  • the interfering RNA can be assembled from two separate oligonucleotides, where one strand is the sense strand and the other is the antisense strand, wherein the antisense and sense strands are self-complementary (i.e., each strand comprises nucleotide sequence that is complementary to nucleotide sequence in the other strand; such as where the antisense strand and sense strand form a duplex or double stranded structure); the antisense strand comprises nucleotide sequence that is complementary to a nucleotide sequence in a target nucleic acid molecule or a portion thereof (i.e., an undesired gene) and the sense strand comprises nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof.
  • interfering RNA is assembled from a single oligonucleotide, where the self-complementary sense and antisense regions are linked by means of nucleic acid based or non-nucleic acid-based linker(s).
  • the interfering RNA can be a polynucleotide with a duplex, asymmetric duplex, hairpin or asymmetric hairpin secondary structure, having self- complementary sense and antisense regions, wherein the antisense region comprises a nucleotide sequence that is complementary to nucleotide sequence in a separate target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof.
  • the interfering can be a circular single-stranded polynucleotide having two or more loop structures and a stem comprising self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof, and wherein the circular polynucleotide can be processed either in vivo or in vitro to generate an active siRNA molecule capable of mediating RNA interference.
  • the interfering RNA coding region encodes a self-complementary RNA molecule having a sense region, an antisense region and a loop region.
  • a self-complementary RNA molecule having a sense region, an antisense region and a loop region.
  • Such an RNA molecule when expressed desirably forms a“hairpin” structure, and is referred to herein as an“shRNA.”
  • the loop region is generally between about 2 and about 10 nucleotides in length. In some embodiments, the loop region is from about 6 to about 9 nucleotides in length.
  • the sense region and the antisense region are between about 15 and about 20 nucleotides in length.
  • the small hairpin RNA is converted into a siRNA by a cleavage event mediated by the enzyme Dicer, which is a member of the RNase III family.
  • Dicer which is a member of the RNase III family.
  • the siRNA is then capable of inhibiting the expression of a gene with which it shares homology.
  • Dicer a member of the RNase III family.
  • siRNAs The target RNA cleavage reaction guided by siRNAs is highly sequence specific.
  • siRNA containing a nucleotide sequences identical to a portion of the target nucleic acid are preferred for inhibition.
  • 100% sequence identity between the siRNA and the target gene is not required to practice the present invention.
  • the invention has the advantage of being able to tolerate sequence variations that might be expected due to genetic mutation, strain polymorphism, or evolutionary divergence.
  • siRNA sequences with insertions, deletions, and single point mutations relative to the target sequence have also been found to be effective for inhibition.
  • siRNA sequences with nucleotide analog substitutions or insertions can be effective for inhibition.
  • the siRNAs must retain specificity for their target, i.e., must not directly bind to, or directly significantly affect expression levels of, transcripts other than the intended target.
  • Trans-cleaving enzymatic nucleic acid molecules can also be used; they have shown promise as therapeutic agents for human disease (Usman & McSwiggen, 1995 Ann. Rep. Med. Chem. 30, 285-294; Christoffersen and Marr, 1995 J. Med. Chem. 38, 2023-2037).
  • Enzymatic nucleic acid molecules can be designed to cleave specific RNA targets within the background of cellular RNA. Such a cleavage event renders the RNA non- functional.
  • enzymatic nucleic acids with RNA cleaving activity act by first binding to a target RNA. Such binding occurs through the target binding portion of a enzymatic nucleic acid which is held in close proximity to an enzymatic portion of the molecule that acts to cleave the target RNA.
  • the enzymatic nucleic acid first recognizes and then binds a target RNA through complementary base pairing, and once bound to the correct site, acts enzymatically to cut the target RNA. Strategic cleavage of such a target RNA will destroy its ability to direct synthesis of an encoded protein. After an enzymatic nucleic acid has bound and cleaved its RNA target, it is released from that RNA to search for another target and can repeatedly bind and cleave new targets.
  • ribozymes that are optimal for catalytic activity would contribute significantly to any strategy that employs RNA- cleaving ribozymes for the purpose of regulating gene expression.
  • the hammerhead ribozyme functions with a catalytic rate (kcat) of about 1 min 1 in the presence of saturating (10 rnM) concentrations of Mg 2+ cofactor.
  • RNA ligase ribozyme has been shown to catalyze the corresponding self-modification reaction with a rate of about 100 min 1 .
  • certain modified hammerhead ribozymes that have substrate binding arms made of DNA catalyze RNA cleavage with multiple turn-over rates that approach 100 min 1 .
  • the inhibitory nucleic acids used in the methods described herein are modified, e.g., comprise one or more modified bonds or bases.
  • a number of modified bases include phosphorothioate, methylphosphonate, peptide nucleic acids, or locked nucleic acid (LNA) molecules.
  • LNA locked nucleic acid
  • Some inhibitory nucleic acids are fully modified, while others are chimeric and contain two or more chemically distinct regions, each made up of at least one nucleotide.
  • inhibitory nucleic acids typically contain at least one region of modified nucleotides that confers one or more beneficial properties (such as, for example, increased nuclease resistance, increased uptake into cells, increased binding affinity for the target) and a region that is a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids.
  • Chimeric inhibitory nucleic acids of the invention may be formed as composite structures of two or more oligonucleotides, modified oligonucleotides, oligonucleosides and/or oligonucleotide mimetics as described above. Such compounds have also been referred to in the art as hybrids or gapmers.
  • the oligonucleotide is a gapmer (contain a central stretch (gap) of DNA monomers sufficiently long to induce RNase H cleavage, flanked by blocks of LNA modified nucleotides; see, e.g., Stanton et al., Nucleic Acid Ther. 2012. 22: 344-359; Nowotny et al., Cell, 121 :1005-1016, 2005; Kurreck, European Journal of Biochemistry 270:1628-1644, 2003; FLuiter et al., Mol Biosyst. 5(8):838-43, 2009).
  • gap central stretch
  • the oligonucleotide is a mixmer (includes alternating short stretches of LNA and DNA; Naguibneva et al., Biomed Pharmacother. 2006 Nov; 60(9):633-8; Orom et al., Gene. 2006 May 10; 372():l37-4l).
  • Representative ETnited States patents that teach the preparation of such hybrid structures comprise, but are not limited to, US patent nos. 5,013,830; 5,149,797; 5, 220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922, each of which is herein incorporated by reference.
  • the inhibitory nucleic acid comprises at least one nucleotide modified at the 2' position of the sugar, most preferably a 2'-0-alkyl, 2'-0-alkyl-0-alkyl or 2'-fluoro- modified nucleotide.
  • RNA modifications include 2'-fluoro, 2'- amino and 2' O-methyl modifications on the ribose of pyrimidines, abasic residues or an inverted base at the 3' end of the RNA.
  • modified oligonucleotides include those comprising modified backbones, for example, phosphorothioates, phosphotri esters, methyl phosphonates, short chain alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages.
  • oligonucleotides with phosphorothioate backbones and those with heteroatom backbones particularly CH2 -NH-0-CH2, CH, ⁇ N(CH3) ⁇ 0 ⁇ CH2 (known as a methylene(methylimino) or MMI backbone], CH2— O— N (CH3)-CH2, CH2 -N (CH3)-N (CH3)-CH2 and O-N (CH3)- CH2 -CH2 backbones, wherein the native phosphodiester backbone is represented as O- P— O- CH,); amide backbones (see De Mesmaeker et al. Ace. Chem. Res.
  • PNA peptide nucleic acid
  • Phosphorus-containing linkages include, but are not limited to, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates comprising 3'alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates comprising 3'-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'; see US patent nos.
  • Morpholino-based oligomeric compounds are described in Dwaine A. Braasch and David R. Corey, Biochemistry, 2002, 41(14), 4503-4510); Genesis, volume 30, issue 3, 2001; Heasman, J., Dev. Biol., 2002, 243, 209-214; Nasevicius et al., Nat. Genet., 2000, 26, 216-220; Lacerra et al., Proc. Natl. Acad. Sci., 2000, 97, 9591-9596; and U.S. Pat. No. 5,034,506, issued Jul. 23, 1991.
  • Cyclohexenyl nucleic acid oligonucleotide mimetics are described in Wang et al., J. Am. Chem. Soc., 2000, 122, 8595-8602.
  • Modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl intemucleoside linkages, mixed heteroatom and alkyl or cycloalkyl intemucleoside linkages, or one or more short chain heteroatomic or heterocyclic intemucleoside linkages.
  • These comprise those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts; see US patent nos.
  • One or more substituted sugar moieties can also be included, e.g., one of the following at the 2' position: OH, SH, SCH 3 , F, OCN, OC3 ⁇ 4 OC3 ⁇ 4, OCH 3 0(CH 2 )n CH 3 , 0(CH 2 )n NH 2 or 0(CH 2 )n CH 3 where n is from 1 to about 10; Ci to C10 lower alkyl, alkoxyalkoxy, substituted lower alkyl, alkaryl or aralkyl; Cl; Br; CN; CF3 ; OCF3; O-, S-, or N-alkyl; O-, S-, or N- alkenyl; SOCH3; S02 CH3; ON02; N02; N3; NH2; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl; an RNA cleaving group; a reporter group; an intercalator;
  • a preferred modification includes 2' -m ethoxy ethoxy [2'- O-CH2CH2OCH3, also known as 2'-0-(2-methoxyethyl)] (Martin et al, Helv. Chim. Acta, 1995, 78, 486).
  • Other preferred modifications include 2'-methoxy (2'-0-CH 3 ), 2'-propoxy (2'-OCH2 CH2CH3) and 2'-fluoro (2'-F).
  • Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3' position of the sugar on the 3' terminal nucleotide and the 5' position of 5' terminal nucleotide.
  • Oligonucleotides may also have sugar mimetics such as cyclobutyls in place of the pentofuranosyl group.
  • Inhibitory nucleic acids can also include, additionally or alternatively, nucleobase (often referred to in the art simply as “base”) modifications or substitutions.
  • nucleobase often referred to in the art simply as “base” modifications or substitutions.
  • “unmodified” or “natural” nucleobases include adenine (A), guanine (G), thymine (T), cytosine (C) and uracil (U).
  • Modified nucleobases include nucleobases found only infrequently or transiently in natural nucleic acids, e.g., hypoxanthine, 6-methyladenine, 5-Me pyrimidines, particularly 5 -methyl cytosine (also referred to as 5-methyl-2' deoxycytosine and often referred to in the art as 5-Me-C), 5-hydroxymethylcytosine (HMC), glycosyl HMC and gentobiosyl HMC, as well as synthetic nucleobases, e.g., 2-aminoadenine, 2- (methylamino)adenine, 2- (imidazolylalkyl)adenine, 2-(aminoalklyamino)adenine or other heterosub stituted alkyladenines, 2-thiouracil, 2-thiothymine, 5-bromouracil, 5- hydroxymethyluracil, 8- azaguanine, 7-deazaguanine, N6 (6-aminohex
  • both a sugar and an internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups.
  • the base units are maintained for hybridization with an appropriate nucleic acid target compound.
  • an oligomeric compound an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA).
  • PNA peptide nucleic acid
  • the sugar -backbone of an oligonucleotide is replaced with an amide containing backbone, for example, an aminoethylglycine backbone.
  • the nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone.
  • PNA compounds comprise, but are not limited to, US patent nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference . Further teaching of PNA compounds can be found in Nielsen et al, Science, 1991, 254, 1497-1500.
  • Inhibitory nucleic acids can also include one or more nucleobase (often referred to in the art simply as “base”) modifications or substitutions.
  • nucleobases comprise the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).
  • Modified nucleobases comprise other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5 -hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2 -propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2- thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudo-uracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8- thioalkyl, 8- hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5- bromo, 5- trifluor
  • nucleobases comprise those disclosed in United States Patent No. 3,687,808, those disclosed in 'The Concise Encyclopedia of Polymer Science And Engineering', pages 858-859, Kroschwitz, J.I., ed. John Wiley & Sons, 1990, those disclosed by Englisch et al., Angewandle Chemie, International Edition', 1991, 30, page 613, and those disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and Applications', pages 289- 302, Crooke, S.T. and Lebleu, B. ea., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention.
  • 5-substituted pyrimidines 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, comprising 2- aminopropyladenine, 5-propynyluracil and 5- propynylcytosine.
  • 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-l .2 ⁇ 0>C (Sanghvi, Y.S., Crooke, S.T. and Lebleu, B., eds, 'Antisense Research and Applications', CRC Press, Boca Raton, 1993, pp. 276-278) and are presently preferred base substitutions, even more particularly when combined with 2'-0-methoxyethyl sugar modifications.
  • nucleobases are described in US patent nos. 3,687,808, as well as 4,845,205; 5,130,302; 5, 134,066; 5, 175, 273; 5, 367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,596,091; 5,614,617; 5,750,692, and 5,681,941, each of which is herein incorporated by reference.
  • the inhibitory nucleic acids are chemically linked to one or more moieties or conjugates that enhance the activity, cellular distribution, or cellular uptake of the oligonucleotide.
  • moieties comprise but are not limited to, lipid moieties such as a cholesterol moiety (Letsinger et ah, Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et ah, Bioorg. Med. Chem. Let., 1994, 4, 1053 -1060), a thioether, e.g., hexyl s tritylthiol (Manoharan et al, Ann. N. Y.
  • Acids Res., 1992, 20, 533-538 an aliphatic chain, e.g., dodecandiol or undecyl residues (Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49- 54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1 ,2-di-O-hexadecyl- rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl.
  • a phospholipid e.g., di-hexadecyl-rac-glycerol or triethylammonium 1 ,2-di-O-hexadecyl- rac-
  • Acids Res., 1990, 18, 3777-3783 a polyamine or a polyethylene glycol chain (Mancharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine or hexylamino-carbonyl- t oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp.
  • conjugate groups of the invention include intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers.
  • Typical conjugate groups include cholesterols, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes.
  • Groups that enhance the pharmacodynamic properties include groups that improve uptake, enhance resistance to degradation, and/or strengthen sequence-specific hybridization with the target nucleic acid.
  • Groups that enhance the pharmacokinetic properties include groups that improve uptake, distribution, metabolism or excretion of the compounds of the present invention. Representative conjugate groups are disclosed in International Patent Application No. PCT/US92/09196, filed Oct. 23, 1992, and U.S. Pat. No. 6,287,860, which are incorporated herein by reference.
  • Conjugate moieties include, but are not limited to, lipid moieties such as a cholesterol moiety, cholic acid, a thioether, e.g., hexyl -5- tritylthiol, a thiochole sterol, an aliphatic chain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac- glycero-3-H-phosphonate, a polyamine or a polyethylene glycol chain, or adamantane acetic acid, a palmityl moiety, or an octadecylamine or hexylamino-carbonyl-oxy cholesterol moiety.
  • lipid moieties such as a cholesterol moiety, cholic acid, a
  • LNAs Locked Nucleic Acids
  • the modified inhibitory nucleic acids used in the methods described herein comprise locked nucleic acid (LNA) molecules, e.g., including [alpha] -L-LN As.
  • LNAs comprise ribonucleic acid analogues wherein the ribose ring is“locked” by a methylene bridge between the 2’-oxgygen and the 4’-carbon - i.e., oligonucleotides containing at least one LNA monomer, that is, one 2'-0,4'-C-methylene- ?-D-ribofuranosyl nucleotide.
  • LNA bases form standard Watson-Crick base pairs but the locked configuration increases the rate and stability of the basepairing reaction (Jepsen et ah, Oligonucleotides, 14, 130-146 (2004)).
  • LNAs also have increased affinity to base pair with RNA as compared to DNA. These properties render LNAs especially useful as probes for fluorescence in situ hybridization (FISH) and comparative genomic hybridization, as knockdown tools for miRNAs, and as antisense oligonucleotides to target mRNAs or other RNAs, e.g., RNAs as described herien.
  • the LNA molecules can include molecules comprising 10-30, e.g., 12-24, e.g., 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in each strand, wherein one of the strands is substantially identical, e.g., at least 80% (or more, e.g., 85%, 90%, 95%, or 100%) identical, e.g., having 3, 2, 1, or 0 mismatched nucleotide(s), to a target region in the RNA.
  • the LNA molecules can be chemically synthesized using methods known in the art.
  • the LNA molecules can be designed using any method known in the art; a number of algorithms are known, and are commercially available (e.g., on the internet, for example at exiqon.com). See, e.g., You et ah, Nuc. Acids. Res. 34:e60 (2006); McTigue et ah, Biochemistry 43 :5388-405 (2004); and Levin et ah, Nuc. Acids. Res. 34:el42 (2006).
  • “gene walk” methods similar to those used to design antisense oligos, can be used to optimize the inhibitory activity of the LNA; for example, a series of oligonucleotides of 10-30 nucleotides spanning the length of a target RNA can be prepared, followed by testing for activity.
  • gaps e.g., of 5-10 nucleotides or more, can be left between the LNAs to reduce the number of oligonucleotides synthesized and tested.
  • GC content is preferably between about 30-60%.
  • General guidelines for designing LNAs are known in the art; for example, LNA sequences will bind very tightly to other LNA sequences, so it is preferable to avoid significant complementarity within an LNA.
  • the LNAs are xylo-LNAs.
  • RNA, cDNA, genomic DNA, vectors, viruses or hybrids thereof can be isolated from a variety of sources, genetically engineered, amplified, and/or expressed/ generated recombinantly.
  • Recombinant nucleic acid sequences can be individually isolated or cloned and tested for a desired activity. Any recombinant expression system can be used, including e.g. in vitro, bacterial, fungal, mammalian, yeast, insect or plant cell expression systems.
  • Nucleic acid sequences of the invention can be inserted into delivery vectors and expressed from transcription units within the vectors.
  • the recombinant vectors can be DNA plasmids or viral vectors.
  • Generation of the vector construct can be accomplished using any suitable genetic engineering techniques well known in the art, including, without limitation, the standard techniques of PCR, oligonucleotide synthesis, restriction endonuclease digestion, ligation, transformation, plasmid purification, and DNA sequencing, for example as described in Sambrook et al. Molecular Cloning: A Laboratory Manual. (1989)), Coffin et al. (Retroviruses. (1997)) and“RNA Viruses: A Practical Approach” (Alan J. Cann, Ed., Oxford University Press, (2000)).
  • Viral vectors comprise a nucleotide sequence having sequences for the production of recombinant virus in a packaging cell.
  • Viral vectors expressing nucleic acids of the invention can be constructed based on viral backbones including, but not limited to, a retrovirus, lentivirus, adenovirus, adeno-associated virus, pox virus or alphavirus.
  • the recombinant vectors capable of expressing the nucleic acids of the invention can be delivered as described herein, and persist in target cells (e.g., stable transformants).
  • Nucleic acid sequences used to practice this invention can be synthesized in vitro by well- known chemical synthesis techniques, as described in, e.g., Adams (1983) J. Am. Chem. Soc. 105:661; Belousov (1997) Nucleic Acids Res. 25:3440-3444; Frenkel (1995) Free Radic. Biol.
  • nucleic acid sequences of the invention can be stabilized against nucleolytic degradation such as by the incorporation of a modification, e.g., a nucleotide modification.
  • nucleic acid sequences of the invention includes a phosphorothioate at least the first, second, or third intemucleotide linkage at the 5' or 3' end of the nucleotide sequence.
  • the nucleic acid sequence can include a 2'-modified nucleotide, e.g., a 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-0-methyl, 2'-0-methoxyethyl (2'-0-M0E), 2'-0-aminopropyl (2'-0-AP), 2'-0- dimethylaminoethyl (2'-0-DMA0E), 2'-0-dimethylaminopropyl (2'-0-DMAP), 2'-0- dimethylaminoethyloxyethyl (2'-0-DMAE0E), or 2'-0— N-methylacetamido (2 -O-NMA).
  • a 2'-modified nucleotide e.g., a 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-0-methyl, 2'-0-methoxyethyl (2'-0-M0E), 2'-0-aminopropyl
  • the nucleic acid sequence can include at least one 2' -O-m ethyl-modified nucleotide, and in some embodiments, all of the nucleotides include a 2'-0-methyl modification.
  • the nucleic acids are“locked,” i.e., comprise nucleic acid analogues in which the ribose ring is“locked” by a methylene bridge connecting the T -O atom and the 4’-C atom (see, e.g., Kaupinnen et ah, Drug Disc. Today 2(3):287-290 (2005); Koshkin et ah, J. Am. Chem. Soc., 120(50): 13252-13253 (1998)).
  • ETS 20100004320, US 20090298916, and US 20090143326 See ETS 20100004320, US 20090298916, and US 20090143326.
  • nucleic acids used to practice this invention such as, e.g., subcloning, labeling probes (e.g., random-primer labeling using Klenow polymerase, nick translation, amplification), sequencing, hybridization and the like are well described in the scientific and patent literature, see, e.g., Sambrook et ah, Molecular Cloning; A Laboratory Manual 3d ed. (2001); Current Protocols in Molecular Biology , Ausubel et ah, eds.
  • labeling probes e.g., random-primer labeling using Klenow polymerase, nick translation, amplification
  • sequencing hybridization and the like
  • the methods described herein can include the administration of pharmaceutical compositions and formulations comprising inhibitors or inhibitory nucleic acid sequences as described herein.
  • the compositions are formulated with a pharmaceutically acceptable carrier.
  • the pharmaceutical compositions and formulations can be administered parenterally, topically, orally or by local administration, such as by aerosol or transdermally.
  • the pharmaceutical compositions can be formulated in any way and can be administered in a variety of unit dosage forms depending upon the condition or disease and the degree of illness, the general medical condition of each patient, the resulting preferred method of administration and the like. Details on techniques for formulation and administration of pharmaceuticals are well described in the scientific and patent literature, see, e.g., Remington: The Science and Practice of Pharmacy, 21 st ed., 2005.
  • the inhibitory nucleic acids can be administered alone or as a component of a pharmaceutical formulation (composition).
  • the compounds may be formulated for administration, in any convenient way for use in human or veterinary medicine.
  • Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.
  • Formulations of the compositions of the invention include those suitable for intradermal, inhalation, oral/ nasal, topical, parenteral, rectal, and/or intravaginal administration.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy.
  • the amount of active ingredient (e.g., nucleic acid sequences of this invention) which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration, e.g., intradermal or inhalation.
  • the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect, e.g., an antigen specific T cell or humoral response.
  • compositions can be prepared according to any method known to the art for the manufacture of pharmaceuticals.
  • Such drugs can contain sweetening agents, flavoring agents, coloring agents and preserving agents.
  • a formulation can be admixtured with nontoxic pharmaceutically acceptable excipients which are suitable for manufacture.
  • Formulations may comprise one or more diluents, emulsifiers, preservatives, buffers, excipients, etc. and may be provided in such forms as liquids, powders, emulsions, lyophilized powders, sprays, creams, lotions, controlled release formulations, tablets, pills, gels, on patches, in implants, etc.
  • compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in appropriate and suitable dosages. Such carriers enable the pharmaceuticals to be formulated in unit dosage forms as tablets, pills, powder, dragees, capsules, liquids, lozenges, gels, syrups, slurries, suspensions, etc., suitable for ingestion by the patient.
  • Pharmaceutical preparations for oral use can be formulated as a solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable additional compounds, if desired, to obtain tablets or dragee cores.
  • Suitable solid excipients are carbohydrate or protein fillers include, e.g., sugars, including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose such as methyl cellulose, hydroxypropylmethyl -cellulose, or sodium carboxy -methyl cellulose; and gums including arabic and tragacanth; and proteins, e.g., gelatin and collagen.
  • Disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate.
  • Push -fit capsules can contain active agents mixed with a filler or binders such as lactose or starches, lubricants such as talc or magnesium stearate, and, optionally, stabilizers.
  • the active agents can be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycol with or without stabilizers.
  • Aqueous suspensions can contain an active agent (e.g., nucleic acid sequences of the invention) in admixture with excipients suitable for the manufacture of aqueous suspensions, e.g., for aqueous intradermal injections.
  • an active agent e.g., nucleic acid sequences of the invention
  • Such excipients include a suspending agent, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia, and dispersing or wetting agents such as a naturally occurring phosphatide (e.g., lecithin), a condensation product of an alkyl ene oxide with a fatty acid (e.g., polyoxyethylene stearate), a condensation product of ethylene oxide with a long chain aliphatic alcohol (e.g., heptadecaethylene oxycetanol), a condensation product of ethylene oxide with a partial ester derived from a fatty acid and a hexitol (e.g., polyoxyethylene sorbitol mono-oleate), or a condensation product of ethylene oxide with a partial ester derived from fatty acid and a hexitol anhydride (e.g., polyoxyethylene sorb
  • the aqueous suspension can also contain one or more preservatives such as ethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents and one or more sweetening agents, such as sucrose, aspartame or saccharin.
  • preservatives such as ethyl or n-propyl p-hydroxybenzoate
  • coloring agents such as a coloring agent
  • flavoring agents such as aqueous suspension
  • sweetening agents such as sucrose, aspartame or saccharin.
  • Formulations can be adjusted for osmolarity.
  • oil-based pharmaceuticals are used for administration of nucleic acid sequences of the invention.
  • Oil-based suspensions can be formulated by suspending an active agent in a vegetable oil, such as arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin; or a mixture of these. See e.g., U.S. Patent No. 5,716,928 describing using essential oils or essential oil components for increasing bioavailability and reducing inter- and intra-individual variability of orally administered hydrophobic pharmaceutical compounds (see also U.S. Patent No. 5,858,401).
  • the oil suspensions can contain a thickening agent, such as beeswax, hard paraffin or cetyl alcohol.
  • Sweetening agents can be added to provide a palatable oral preparation, such as glycerol, sorbitol or sucrose. These formulations can be preserved by the addition of an antioxidant such as ascorbic acid.
  • an injectable oil vehicle see Minto (1997) J. Pharmacol. Exp. Ther. 281 :93-102.
  • Pharmaceutical formulations can also be in the form of oil-in-water emulsions.
  • the oily phase can be a vegetable oil or a mineral oil, described above, or a mixture of these.
  • Suitable emulsifying agents include naturally-occurring gums, such as gum acacia and gum tragacanth, naturally occurring phosphatides, such as soybean lecithin, esters or partial esters derived from fatty acids and hexitol anhydrides, such as sorbitan mono-oleate, and condensation products of these partial esters with ethylene oxide, such as polyoxyethylene sorbitan mono-oleate.
  • the emulsion can also contain sweetening agents and flavoring agents, as in the formulation of syrups and elixirs. Such formulations can also contain a demulcent, a preservative, or a coloring agent.
  • these injectable oil-in-water emulsions of the invention comprise a paraffin oil, a sorbitan monooleate, an ethoxylated sorbitan monooleate and/or an ethoxylated sorbitan trioleate.
  • the pharmaceutical compounds can also be administered by in intranasal, intraocular and intravaginal routes including suppositories, insufflation, powders and aerosol formulations (for examples of steroid inhalants, see e.g., Rohatagi (1995) J. Clin. Pharmacol. 35:1187-1193; Tjwa (1995) Ann. Allergy Asthma Immunol. 75:107-111).
  • Suppositories formulations can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at body temperatures and will therefore melt in the body to release the drug.
  • suitable non-irritating excipient which is solid at ordinary temperatures but liquid at body temperatures and will therefore melt in the body to release the drug.
  • Such materials are cocoa butter and polyethylene glycols.
  • the pharmaceutical compounds can be delivered topically or transdermally, by a topical route, formulated as applicator sticks, solutions, suspensions, emulsions, gels, creams, ointments, pastes, jellies, paints, powders, and aerosols.
  • compositions for transdermal application can further comprise cosmetically-acceptable carriers or vehicles and any optional components.
  • cosmetically acceptable carriers, vehicles and optional components are known in the art and include carriers and vehicles suitable for application to skin (e.g., sunscreens, creams, milks, lotions, masks, serums, etc.), see, e.g., U.S. Patent Nos. 6,645,512 and 6,641,824.
  • optional components that may be desirable include, but are not limited to absorbents, anti -acne actives, anti-caking agents, anti -cellulite agents, anti-foaming agents, anti-fungal actives, anti inflammatory actives, anti -microbial actives, anti -oxidants, antiperspirant/deodorant actives, anti-skin atrophy actives, anti-viral agents, anti-wrinkle actives, artificial tanning agents and accelerators, astringents, barrier repair agents, binders, buffering agents, bulking agents, chelating agents, colorants, dyes, enzymes, essential oils, film formers, flavors, fragrances, humectants, hydrocolloids, light diffusers, nail enamels, opacifying agents, optical brighteners, optical modifiers, particulates, perfumes, pH adjusters, sequestering agents, skin conditioners/moisturizers, skin feel modifiers, skin protectants, skin sensates, skin treating agents, skin exfoliating agents,
  • the pharmaceutical compounds can also be delivered as microspheres for slow release in the body.
  • microspheres can be administered via intradermal injection of drug which slowly release subcutaneously; see Rao (1995) J. Biomater Sci. Polym. Ed. 7:623-645; as biodegradable and injectable gel formulations, see, e.g., Gao (1995) Pharm. Res. 12:857-863 (1995); or, as microspheres for oral administration, see, e.g., Eyles (1997) J. Pharm. Pharmacol. 49:669-674.
  • the pharmaceutical compounds can be parenterally administered, such as by intravenous (IV) administration or administration into a body cavity or lumen of an organ.
  • IV intravenous
  • These formulations can comprise a solution of active agent dissolved in a pharmaceutically acceptable carrier.
  • Acceptable vehicles and solvents that can be employed are water and Ringer's solution, an isotonic sodium chloride.
  • sterile fixed oils can be employed as a solvent or suspending medium.
  • any bland fixed oil can be employed including synthetic mono- or diglycerides.
  • fatty acids such as oleic acid can likewise be used in the preparation of injectables. These solutions are sterile and generally free of undesirable matter.
  • These formulations may be sterilized by conventional, well known sterilization techniques.
  • the formulations may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents, e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like.
  • concentration of active agent in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight, and the like, in accordance with the particular mode of administration selected and the patient's needs.
  • the formulation can be a sterile injectable preparation, such as a sterile injectable aqueous or oleaginous suspension. This suspension can be formulated using those suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation can also be a suspension in a nontoxic parenterally- acceptable diluent or solvent, such as a solution of l,3-butanediol.
  • the administration can be by bolus or continuous infusion (e.g., substantially uninterrupted introduction into a blood vessel for a specified period of time).
  • the pharmaceutical compounds and formulations can be lyophilized.
  • Stable lyophilized formulations comprising an inhibitory nucleic acid can be made by lyophilizing a solution comprising a pharmaceutical of the invention and a bulking agent, e.g., mannitol, trehalose, raffmose, and sucrose or mixtures thereof.
  • a process for preparing a stable lyophilized formulation can include lyophilizing a solution about 2.5 mg/mL protein, about 15 mg/mL sucrose, about 19 mg/mL NaCl, and a sodium citrate buffer having a pH greater than 5.5 but less than 6.5. See, e.g., U.S. 20040028670.
  • compositions and formulations can be delivered by the use of liposomes.
  • liposomes particularly where the liposome surface carries ligands specific for target cells, or are otherwise preferentially directed to a specific organ, one can focus the delivery of the active agent into target cells in vivo. See, e.g., U.S. Patent Nos. 6,063,400; 6,007,839; Al-Muhammed (1996) J. Microencapsul. 13 :293-306; Chonn (1995) Curr. Opin. Biotechnol. 6:698-708; Ostro (1989) Am. J. Hosp. Pharm. 46: 1576-1587.
  • liposome means a vesicle composed of amphiphilic lipids arranged in a bilayer or bilayers. Liposomes are unilamellar or multilamellar vesicles that have a membrane formed from a lipophilic material and an aqueous interior that contains the composition to be delivered. Cationic liposomes are positively charged liposomes that are believed to interact with negatively charged DNA molecules to form a stable complex. Liposomes that are pH-sensitive or negatively-charged are believed to entrap DNA rather than complex with it. Both cationic and noncationic liposomes have been used to deliver DNA to cells.
  • Liposomes can also include "sterically stabilized" liposomes, i.e., liposomes comprising one or more specialized lipids. When incorporated into liposomes, these specialized lipids result in liposomes with enhanced circulation lifetimes relative to liposomes lacking such specialized lipids.
  • sterically stabilized liposomes are those in which part of the vesicle forming lipid portion of the liposome comprises one or more glycolipids or is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety.
  • PEG polyethylene glycol
  • compositions of the invention can be administered for prophylactic and/or therapeutic treatments.
  • compositions are administered to a subject who is need of reduced triglyceride levels, or who is at risk of or has a disorder described herein, in an amount sufficient to cure, alleviate or partially arrest the clinical manifestations of the disorder or its complications; this can be called a therapeutically effective amount.
  • pharmaceutical compositions of the invention are administered in an amount sufficient to decrease levels of TRM in the tissue.
  • the amount of pharmaceutical composition adequate to accomplish this is a therapeutically effective dose.
  • the dosage schedule and amounts effective for this use i.e., the dosing regimen, will depend upon a variety of factors, including the stage of the disease or condition, the severity of the disease or condition, the general state of the patient's health, the patient’s physical status, age and the like. In calculating the dosage regimen for a patient, the mode of administration also is taken into consideration.
  • the dosage regimen also takes into consideration pharmacokinetics parameters well known in the art, i.e., the active agents’ rate of absorption, bioavailability, metabolism, clearance, and the like (see, e.g., Hidalgo-Aragones (1996) J. Steroid Biochem. Mol. Biol. 58:611 -617; Groning (1996) Pharmazie 51 :337-341; Fotherby (1996) Contraception 54:59-69; Johnson (1995) J. Pharm. Sci. 84:1144-1146; Rohatagi (1995) Pharmazie 50:610-613; Brophy (1983) Eur. J. Clin. Pharmacol. 24:103-108; Remington: The Science and Practice of Pharmacy, 21 st ed., 2005).
  • the active agents rate of absorption, bioavailability, metabolism, clearance, and the like
  • formulations can be given depending on for example: the dosage and frequency as required and tolerated by the patient, the degree and amount of therapeutic effect generated after each administration (e.g., effect on tumor size or growth), and the like.
  • the formulations should provide a sufficient quantity of active agent to effectively treat, prevent or ameliorate conditions, diseases or symptoms.
  • pharmaceutical formulations for oral administration are in a daily amount of between about 1 to 100 or more mg per kilogram of body weight per day.
  • Lower dosages can be used, in contrast to administration orally, into the blood stream, into a body cavity or into a lumen of an organ.
  • Substantially higher dosages can be used in topical or oral administration or administering by powders, spray or inhalation.
  • Actual methods for preparing parenterally or non-parenterally administrable formulations will be known or apparent to those skilled in the art and are described in more detail in such publications as Remington: The Science and Practice of Pharmacy, 21 st ed., 2005.
  • Various studies have reported successful mammalian dosing using complementary nucleic acid sequences.
  • LNAs locked nucleic acids
  • the methods described herein can include co-administration with other drugs or pharmaceuticals, e.g., compositions for providing cholesterol homeostasis.
  • the inhibitory nucleic acids can be co-administered with drugs for treating or reducing risk of a disorder described herein.
  • an“effective amount” is an amount sufficient to effect beneficial or desired results.
  • a therapeutic amount is one that achieves the desired therapeutic effect. This amount can be the same or different from a prophylactically effective amount, which is an amount necessary to prevent onset of disease or disease symptoms.
  • An effective amount can be administered in one or more administrations, applications or dosages.
  • a therapeutically effective amount of a therapeutic compound i.e., an effective dosage
  • the compositions can be administered one from one or more times per day to one or more times per week; including once every other day.
  • treatment of a subject with a therapeutically effective amount of the therapeutic compounds described herein can include a single treatment or a series of treatments.
  • Dosage, toxicity and therapeutic efficacy of the therapeutic compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50.
  • Compounds which exhibit high therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
  • the data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture.
  • IC50 i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms
  • levels in plasma may be measured, for example, by high performance liquid chromatography.
  • WT Wide-type C57BL/6, CD45.l + , Thyl.l + , Ragl _/_ and mMT mice were purchased from Jackson Laboratory. Thyl.l + Ragl _/_ OT-I mice were maintained through routine breeding in the animal facility of Harvard Institute of Medicine, Harvard Medical School. Fabp4 ' Fabp5 ⁇ and Fabp4/5 dKO mice were kindly provided by Gokhan S. Hotamisligil (Harvard T.H. Chan School of Public Health). Mice were bred to generate Thyl.l + CD45. l + WT; Thyl. l + CD45.2 + Fabp4/5 dKO; Thyl.
  • mice were performed in accordance with the guidelines put forth by the Center for Animal Resources and Comparative Medicine at Harvard Medical School, and all protocols and experimental plans were approved by the HMS IACUC in advance of their performance. Mice were randomly assigned to each group before start and experiments were performed blinded with respect to treatment. For survival experiments, mice that had lost over 25% of original BW were euthanized.
  • VACV Recombinant VACV expressing OT-I T cell epitope Ova 25 7-264 and Western Reserve strain (WR-VACV) were originally obtained from B. Moss (NIH). Virus was expanded and titered by standard procedures as described previously 9 . 2 c 10 6 p.f.u. of VAC VOVA was used for infection by either skin scarification or intra-tracheal infection. 2 x 10 6 p.f.u. WR-VACV was used at a lethal dose in intranasal infections, as described previously 9 .
  • anti-mouse antibodies were obtained from BD PharMingen: PerCP-conjugated anti-CD3e (553067), PE-conjugated anti-CD8 (557654), PE-Cy7-conjugated anti-CD8 (552877), APC-Cy 7 -conjugated anti-CD8 (557654), PE-conjugated anti-Thyl. l (561404), APC -conjugated anti-Thyl.
  • Biolegend Alexa Fluor 488-conjugated anti-CD3e (100321), Alexa Fluor 647-conjugated anti-CD4 (100424), Alexa Fluor 594-conjugated anti-Ep-CAM (118222), FITC-conjugated anti-CD45.1 (110706), PE-conjugated anti-CD45.l (110708), PE-Cy7-conjugated anti-CD45.2 (109830), APC- conjugated anti-CD45.2 (109814), PE-conjugated anti-CD45.2 (109808), APC -conjugated anti-KLRGl (138412), PE-conjugated anti-CD44 (103008), PE-Cy7-conjugated anti-CD44 (103030), PE-Cy 7-conjugated anti-CD69 (104512), APC -conjugated anti-CDl03 (121414), APC -conjugated anti-integrin a4b7 (120608).
  • APC-Cy 7-conjugated anti-CD3 (300425), PerCp-conjugated anti- CD4 (317431), APC-conjugated anti-CD8 (300911), Alexa Fluor 488-conjugated anti-CD69 (310916), PE-Cy 7-conjugated anti-CD69 (310911), PE-conjugated anti-CD62L (304805),
  • FITC-conjugated anti-CD45RO (304204).
  • Abeam anti-human FABP4 (9B8D).
  • NOVTJS Alexa Fluor 405 -conjugated anti-human FABP5 (FAB3077V).
  • PE-conjugated B8R20-27/H- 2Kb pentamers were obtained from Prolmmune Ltd, and stained according to the manufacturer’s protocol.
  • E- or P-selectin ligand expression was examined by incubating cells with rmE-Selectin/Fc Chimera (575-ES; R&D System) or rmP-Selectin/Fc Chimera (737-PS; R&D System) in conjunction with PerCP -conjugated F(ab')2 fragments of goat anti -human IgG F(c) antibody (109-126-170; Jackson ImmunoRe search).
  • Bodipy conjugated palmitate Bodipy FL C16; D-3821; Thermo Fisher
  • Lymph nodes and spleen were harvested and pressed through a 70-pm nylon cell strainer to prepare cell suspensions.
  • Red blood cells (RBC) were lysed using RBC lysis buffer (00-4333- 57; eBioscience). Skin tissue was excised after hair removal, separated into dorsal and ventral halves, minced, and then incubated in Hanks balanced salt solution (HBSS) supplemented with 1 mg/ml collagenase A (11088785103; Roche) and 40 pg/ml DNase I (10104159001; Roche) at 37 °C for 30 min. After filtration through a 70-pm nylon cell strainer, cells were collected and washed three times with cold PBS before staining.
  • HBSS Hanks balanced salt solution
  • T cells were purified by magnetic cell sorting using a mouse CD8a + T-cell isolation kit (130-104-075; Miltenyi Biotec) or a mouse CD4 + T-cell isolation kit (130-104-454; Miltenyi Biotec), according to the manufacturer’s protocols. T cells were then transferred intravenously into female recipient mice at a total number of 5 c 10 5 or 2.5 c 10 5 cells/population in co-transfer experiments where cell types were transferred at a ratio of 1 : 1. To generate mixed bone marrow chimeras, T cells- and NK cell-depleted Thyl.l + CD45. l + WT and Thyl.
  • mice were rested for eight weeks before infection for full reconstitution of T cells and restoration of an intact immune system.
  • Ragl /_ T-cell reconstituted mice were generated by adoptive transfer of 3.5 c 10 6 CD4 + with 2 10 6 CD8 + WT or CD8 + Fabp4/5 dKO cells.
  • T cells were labeled with carboxyfluorescein succinimidyl ester (CFSE, 65-0850; eBioscience) before co-transfer, where indicated.
  • CFSE carboxyfluorescein succinimidyl ester
  • mice were treated daily with FTY720 (10006292; CAYMAN, 1 mg/kg) by intraperitoneal injection or with etomoxir (E1905; Sigma-Aldrich, 1 pg/site), GW9662 (M6191, Sigma- Aldrich, 1 mg/kg) or trimetazidine (653322, Sigma-Aldrich, 1 mg/kg) by intradermal injection.
  • FTY720 10006292; CAYMAN, 1 mg/kg
  • etomoxir E1905; Sigma-Aldrich, 1 pg/site
  • GW9662 M6191, Sigma- Aldrich, 1 mg/kg
  • trimetazidine 653322, Sigma-Aldrich, 1 mg/kg
  • OT -I cells from 15-20 mice were sorted with a FACSAria
  • RNA was prepared as described above.
  • Bio-Rad iCycler iQ Real-Time PCR Detection System Bio-Rad was used with the following settings: 45 cycles of 15 s of denaturation at 95 °C, and 1 min of primer annealing and elongation at 60 °C.
  • mouse Fabp4 forward (5 -TTT CCT TCA AAC TGG GCG TG-3') and mouse Fabp4 reverse (5 '-CAT TCC ACC ACC AGC TTG TC-3'); mouse Fabp5 forward (5'- AAC CGA GAG CAC AGT GAA G-3 ') and mouse Fabp5 reverse (5 '-ACA CTC CAC GAT CAT CTT CC-3 '); mouse Pparj forward (5 '-TCG CTG ATG CAC TGC CTA TG-3 ') and mouse Pparj reverse (5 '-GAG AGG TCC AC A GAG CTG ATT- 3'); mouse b-actin forward (5 '-CAT TGC TGA CAG GAT GCA GAA GG-3') and mouse b- actin reverse (5'-
  • each standard curve was constructed using lO-fold serial dilutions of target gene template ranging from 10 7 to 10 2 copies per mL and obtained by plotting values of the logarithm of their initial template copy numbers versus the mean Ct values.
  • the actual copy numbers of target genes were determine by relating the Ct value to a standard curve.
  • mice were perfused with buffer A (0.2 M NaH 2 P0 4 , 0.2M Na 2 HP0 4 , 0.2 M L-lysine and 0.1 M sodium periodate with 2% paraformaldehyde) and infected skin sites were harvested and incubated for 30 min on ice in buffer A. Skin tissue was washed twice with PBS and incubated for 30 min at 4 °C in 20% sucrose. Fixed tissue was embedded in OCT (Tissue Tek IA018; Sakura) and frozen in liquid nitrogen. Skin sections were performed on a cryostat (Leica CM1850 UV) at 6-pm thickness and air-dried for 6-8 h.
  • buffer A 0.2 M NaH 2 P0 4 , 0.2M Na 2 HP0 4 , 0.2 M L-lysine and 0.1 M sodium periodate with 2% paraformaldehyde
  • Sections were then fixed in -20 °C acetone for 5 min, rehydrated with PBS, and blocked with 2% FCS in PBS for 15 min at room temperature (20 °C). Sections were stained with rabbit anti-mouse/human FABP4 antibody (EPR3579; ab9250l, Abeam), rabbit anti-mouse/human FABP5 (H-45, sc-50379, Santa Cruz) overnight at 4 °C in a semi-humid chamber. Sections were rinsed for 10 min in PBS, and labeled with donkey anti-rabbit Rhodamine RedTM-X (711-296-152; Jackson ImmunoResearch) for 1 hr at room temperature (20 °C).
  • donkey anti-rabbit Rhodamine RedTM-X 711-296-152; Jackson ImmunoResearch
  • Sections were rinsed for 10 min in PBS, and stained with Alexa Fluor647-conjugated anti -mouse Thy 1.1 (202508, Biolegend) in PBS for 1 hr at room temperature. Then sections were rinsed three times (for 5 min each time) with TBS-Tween 20 by shaking and mounted with ProLong Diamond Antifade Mountant with DAPI (P36962; ThermoFisher). For tissue lipid visualization, sections were stained with BODIPY® 493/503 (4,4-Difluoro-l,3,5,7,8-Pentamethyl-4-Bora-3a,4a-Diaza-s-Indacene) (D3922, Molecular Probes) before mounted. Images were acquired with Leica TCS SP8 confocal microscopy (Harvard NeuroDiscovery Center Optical Imaging Core) and analyzed with ImageJ.
  • Scrambled, Ppary and Cptla siRNA GFP lentiviruses were purchased from ABM (Applied Biological Materials Inc., Canada) with sequences as following: scrambled siRNA: GGG TGA ACT C AC GTC AGA A; Ppary KD 1 : AAT ATG ACC TGA AGC TCC AAG AAT A; Ppary
  • KD2 GTC TGC TGA TCT GCG AGC C; Cptla KDl : GGA GCG ACT CTT CAA TAC TTC CCG CAT CC, Cptla KD2: GGT CAT AGA GAC ATC CCT AAG CAG TGC CA.
  • OT-I mice were infected with 2 x 10 6 VACVOVA by skin scarification.
  • CD8 + T cells were harvested from draining lymph nodes and incubated in medium with 10 pg/ml polybrene and 20 ng/ml hIL-2 at 37 °C for 30 min.
  • mice were sacrificed and the number of siRNA transduced OT - I cells in the infected skin tissue were isolated and enumerated by flow cytometry based on the GFP marker.
  • Recipient and both donor populations used for co-transfers differed in CD90 and CD45 alleles (being CD90.2/45.2, CD90.1/45.1 or CD90.1/45.2 - the combinations differing between experiments) to allow for identification of donor populations.
  • Oxidation of exogenous free fatty acids was measured using XF Palmitate-BSA FAO substrate with XF cell mito stress kit according to the manufacturer’s protocol (Seahorse Bioscience).
  • Freshly isolated and sorted T cells (2.5 x 10 5 ) were incubated for 30 min with FAO assay medium (111 mM NaCl, 4.7 mM KC1, 1.25 mM CaCl 2 , 2.0 mM MgS0 4 , 1.2 mM Na 2 HP0 4 , 2.5 mM glucose, 0.5 mM carnitine and 5 mM HEPES).
  • FAO assay medium 111 mM NaCl, 4.7 mM KC1, 1.25 mM CaCl 2 , 2.0 mM MgS0 4 , 1.2 mM Na 2 HP0 4 , 2.5 mM glucose, 0.5 mM carnitine and 5 mM HEPES.
  • etomoxir 40 mM
  • BSA 34 pM
  • palmitate-BSA 200 pM palmitate conjugated with 34 pM BSA
  • OCR oxygen- consumption rate
  • VACV load was evaluated by quantitative real-time PCR as described previously 3 .
  • inoculated skin samples were harvested and DNA was purified with the DNeasy Mini Kit (51304; Qiagen) according to the manufacturer's protocol.
  • Real-time PCR was performed with the Bio-Rad iCycler iQ Real-Time PCR Detection System (Bio-Rad Laboratories).
  • the primers and TaqMan probe used in the quantitative PCR assay are specific for the ribonucleotide reductase Vvl4L of VACV.
  • sequences are (forward) 5'-GAC ACT CTG GCA GCC GAA AT-3'; (reverse) 5'-CTG GCG GCT AGA ATG GCA TA-3'; (probe) 5 AGC AGC CAC TTG TAC TAC AC A AC A TCC GGA-3
  • the probe was 5 '-labeled with FAM and 3 '-labeled with TAMRA (Applied Biosystems, Foster City, CA).
  • Amplification reactions were performed in a 96-well PCR plate (Bio-Rad Laboratory) in a 20 pl volume containing 2x TaqMan Master Mix (Applied Biosystems), 500 nM forward primer, 500 nM reverse primer, 150 nM probe, and the template DNA.
  • Thermal cycling conditions were 50°C for 2 min and 95°C for 10 min for one cycle, followed by 45 cycles of amplification (94°C for 15 s and 60°C for 1 min).
  • a standard curve was established from DNA of a VACV stock with previously determined titer.
  • Corresponding CT values obtained by the real-time PCR methods were plotted on the standard curve to estimate viral load in the skin samples.
  • Infected skin was harvested 6 days post VACVOVA re-infection and single cell suspensions were prepared as described above. Then cells were incubated with 2 pg/ml SINFEKL peptide of ovalbumin (RP 10611; GenScript) in the presence of Brefeldin A (00-4506-51; eBioscience) for 7 hr. Fc receptors were blocked with CD16/CD32 monoclonal antibodies (14-0161-82; eBioscience).
  • IFN-g 554413; BD
  • IFN-g isotype control 554686; BD
  • Comparisons for two groups were calculated using Student’s t test (two tailed). Comparisons for more than two groups were calculated with one-way analysis of variance (ANOVA) followed by Bonferroni’s multiple comparison tests. Two-way ANOVA with Holm- Bonferroni post hoc analysis was used to compare weight loss between groups and Log-rank (Mantel-Cox) test was used for survival curves p ⁇ 0.05 was considered statistically significant.
  • Bodipy conjugated palmitate Bodipy FL C16; D-3821; Thermo Fisher
  • Bodipy FL C16 Bodipy FL C16
  • D-3821 Thermo Fisher
  • Bodipy uptake was quenched by adding 4x volume of ice-cold PBS with 2% FBS and then cells washed twice prior to flow cytometry analysis. Annexin V staining was included to exclude dead/dying cells during FACS data acquisition.
  • P0500 pM palmitic acid
  • Example 1 Survival of tissue-resident memory T cells requires exogenous lipid uptake and metabolism
  • OT-I transgenic T cells were transferred into recipient mice one day before immunization with a rVACVov A 14 .
  • OT-I cells were readily found in the skin at day 5 post infection and reached their maximal level at day 10 before beginning to decrease in numbers (Fig. 5A). Skin-infiltrating OT-I cells were sorted at different timepoints after infection and were analyzed by transcriptional profiling.
  • Peroxisome proliferator-activated receptors are adipogenic regulators that have been reported to influence Fabp4 and Fabp5 gene expression 16 .
  • Ppary but not Ppara or Ppar®, was selectively up-regulated in TRM compared to TN, TCM and TEM (Fig. IF).
  • Knockdown of Ppary expression using lentiviral siRNA, or treatment of mice with GW9662 (an irreversible PPAR antagonist), inhibited Fabp4 and Fabp5 gene expression in CD8 + TRM Fig. 1G, Fig. 5E-F.
  • naive T cells Upon activation, naive T cells undergo metabolic reprogramming as they proliferate and develop into different subsets of memory T cells 17 18 .
  • the strongly upregulated TRM genes Fabp4 and Fabp5 encode for lipid chaperone proteins that bind to hydrophobic ligands to coordinate lipid uptake and intracellular trafficking 19 .
  • Extracellular free fatty acid (FFA) could be visualized in mouse epidermis, where skin CD8 + TRM localize 3 . Given the magnitude of their upregulation, we hypothesized that FABP4 and FABP5 might play a role in CD8 + TRM physiology in skin. To test our hypothesis, we first compared the extracellular FFA uptake of OT-I memory T cell subtypes in vitro.
  • OT-I WT and OT-l Fabp4/5 dKO cells were mixed at 1 :1 ratio and transferred into congenic recipients. Mice were then infected with VACVOVA, and the number of OT-I WT and OT-I Fabp4/5 dKO cells in different anatomic compartments was assessed. No difference was observed between the number of spleen WT OT-I and OT-I Fabp4/5 dKO TCM at any timepoint, indicating that deficiency of FABP4 and FABP5 did not affect TCM survival (Fig. 2C).
  • OT-I Fabp4/5 dKO cells in skin displayed a profound defect in persistence, beginning 25 days post infection.
  • the ratio of OT-I Fabp4/5 dKO/OT-l WT TRM cells declined steadily over time thereafter (Fig. 2C).
  • Deficiency of both FABP4 and FABP5 also decreased numbers of OT-I TRM detectable by IF without affecting their recruitment or tissue localization (Fig. 2D, Fig. 8).
  • Fabp5 _/ , or Fabp4 +I ⁇ Fabp 5 +/ TRM showed no defect in long-term survival (Figs. 6A-D), consistent with the compensatory and redundant role of these two molecules 20 .
  • OT-I effector T cells acquired more FFA compared to TN, TCM and TEM but less FFA compared to TRM (Fig. 7A-B).
  • OT-I Fabp4/5 dKO T eff displayed a similar proliferative capacity and tissue-homing receptor expression as WT T e ff at 60 hr post infection (Fig. 7C).
  • TCM or Fabp4/5 dKO TRM OT-I cells did not demonstrate an increased OCR when supplied with exogenous fatty acid, and the addition of etomoxir had no effect on their cellular respiration (Fig. 2F).
  • Fig. 2G, Figs. 11A-C In vivo knockdown of Cptla or treatment of mice with either etomoxir or trimetazidine 23 decreased the number of OT-I WT TRM to an extent similar to their Fabp4/5 dKO counterparts (Fig. 2G, Figs. 11A-C).
  • T eff early after infection, roughly equivalent numbers of WT and Fabp4/5 dKO CD8 + effector T cells (T eff ) were found in skin (Fig. 2C). Compared to skin TRM isolated at day 30, skin infiltrating T eff isolated at days 10 and 15 displayed a lower OCR but a higher basal extracellular acidification rate (ECAR), which corresponds to glycolysis 21 (Fig. 11D). Deficiency in Fabp4/5 decreased the OCR of TRM at day 30 post infection but had no effect on the ECAR of skin infiltrating T eff (Fig. 11D). These data suggest that early skin infiltrating T eff utilize glycolysis, which is unaffected by Fabp4/5 gene expression.
  • ECAR extracellular acidification rate
  • TRM are more effective than are TCM at clearing tissue VACV infections 3 .
  • FTY720 a sphingosine-l -phosphate receptor antagonist, was injected into mice to assess the contribution of circulating TCM to viral clearance (Fig. 12A).
  • Established OT-I WT TRM were highly effective at clearing virus from skin, which was rapid and unaffected by FTY720 treatment (Fig.
  • OT-I TRM lacking Fabp4 and Fabp5 were less effective at viral clearance; this was exaggerated following FTY720 treatment (Fig. 3B).
  • Treatment of mice with etomoxir reduced the VACV clearing capacity of OT-I WT TRM to a level comparable to that of their Fabp4/5 dKO counterparts (Fig. 3B), suggesting dependence upon oxidative metabolism of FFA.
  • OT-I I'abp4 5 dKO TRM displayed impaired IFN-g production compared to WT counterparts (Fig. 3C-D). Similar results were obtained for OT-I Fabp4/5 dKO TRM residing at skin sites distant from the infection (Figs. 12B-C).
  • mice with Fabp4/5 dKO CD8 + TRM showed marked weight loss after challenge and were only partially protected from lethality (Fig. 3F-G).
  • lung Fabp4/5 dKO CD8 + TRM were less protective against lethal respiratory VACV infection, and required the recruitment of circulating TCM.
  • WT CD8 + TRM alone protected 50% of mice from this overwhelming lethal VACV infection, consistent with our previous data 9 .
  • TRM in human skin have been implicated in the pathogenesis of several human skin diseases, including psoriasis 10,24 ’ 25 .
  • psoriasis 10,24 psoriasis 10,24 ’ 25 .
  • FABP4 and FABP5 were both strongly expressed in human skin CD8 + TRM compared to human blood TN, TCM and TEM by FACS analysis (Fig. 4A-B).
  • Psoriasis is a chronic and recurring autoimmune disease (Fig. 4C) thought to be mediated by CD8 + TRM cells 5,26 .
  • Fig. 4C a chronic and recurring autoimmune disease
  • Lipids could be visualized in lesional scalp skin and readily detectable FABP4 and FABP5 protein expression could be detected in human psoriatic skin CD8 + TRM.
  • Incubation in vitro with exogenous Bodipy C16 showed that human skin CD8 + TRM internalized more exogenous FFA compared to blood TN, TCM and TEM (Fig. 4D) suggesting a similar role of FABP4 and FABP5 in fatty acid uptake of human CD8 + TRM as we demonstrated in mice.
  • TCM Skin and other epithelial tissues are lipid-rich but nutrient-poor microenvironments 15,27 , and CD8 + TRM appear to utilize mitochondrial ® oxidation of exogenous FFA or other lipids to support both their longevity and protective function.
  • TCM depend in part on FAO for cellular metabolism 17,28 , our data show that TCM cannot effectively internalize exogenous FFA.
  • Cell-intrinsic lipolysis and increased glycerol transport are used by TCM to support metabolic programming necessary for development 17,28,29 , but the dependence upon exogenous FFA uptake and metabolism for long-term survival is unique to TRM.
  • mice injected intradermally with etomoxir and mice with Cptla knockdown in OT-I cells suggest that the etomoxir effects on CD8 + TRM persistence were mediated through CPT1A 30 .
  • generation of long-lived TRM are a goal of effective vaccination 4
  • dysfunction of TRM underlies many auto-inflammatory tissue disorders 4,5
  • a more detailed understanding of the unique lipid metabolic programs intrinsic to TRM and how these programs might be manipulated to increase or decrease TRM longevity and function will be a subject of future investigation.
  • OT-l skin TRM were compared to Cd36-/- OT-l skin TRM at day 30 in the uptaking of extracellular Bodipy conjugated palmitate.
  • Cd36-/- TRM internalize BiodipyFLl6 less efficiently than do WT OT-l TRM. This uptake is specific, and could be blocked with unlabeled palmitate (Fig 13 A).
  • We next performed experiments mixing equal numbers of WT OT-l cells and cd36-/- OT-l cells, transferring into naive recipient mice, and then infecting the mouse with VACVOVA.
  • TCM from spleen and T cells from skin were harvested at various time points, and the ratio of WT to cd36-/- OT-l cells was assessed by flow cytometry. No difference could be observed between cd36-/- and WT TCM over the 90 days of the experiment (Fig 13B). However, in skin beginning at day 45, there was a survival disadvantage of cd36-/- TRM, which gradually increased through day 90 (Fig 13B).
  • C57BL/6 mice were sensitized by topical applications of 20 pl of 0.25% DNFB diluted in acetone olive oil (3:1 v/v) at the indicated time points (day 0, 1 and 7).
  • Challenge was performed at the infection site with 20 m ⁇ of DNFB -acetone olive oil (aOO) (0.25%), with or without etomoxir (1 ug/site) and trimetazidine (10 ug/site), at the indicated time points. Then the ear thickness of the mice was measured with a digital thickness gauge (Mitsutoyo) for the following days.

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Abstract

L'invention concerne des méthodes de traitement ou de réduction du risque de développement ou de progression d'une maladie médiée par des lymphocytes T mémoires résidant dans les tissus (TRM) comprenant l'administration d'une quantité thérapeutiquement efficace d'un ou de plusieurs inhibiteurs de l'absorption de lipides exogènes et d'acides gras libres ou de l'oxydation bêta mitochondriale des FFA exogènes internalisés (par exemple, des inhibiteurs de CD36 et/ou des antagonistes de FABP, par exemple, des inhibiteurs de FABP4 et/ou de FABP5, et/ou CPT1) à un sujet en ayant besoin.
PCT/US2019/018341 2018-02-19 2019-02-15 Ciblage du métabolisme des lipides et de l'oxydation des acides gras libres (ffa) pour traiter des maladies médiées par des lymphocytes t mémoires résidents (trm) WO2019161294A1 (fr)

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US20140363522A1 (en) * 2011-03-17 2014-12-11 The University Of Chicago Compositions and Methods for Treating and/or Preventing Cancer by Inhibiting Fatty Acid Binding Proteins
US20160319003A1 (en) * 2015-04-30 2016-11-03 President And Fellows Of Harvard College Anti-aP2 Antibodies and Antigen Binding Agents to Treat Metabolic Disorders

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US20140363522A1 (en) * 2011-03-17 2014-12-11 The University Of Chicago Compositions and Methods for Treating and/or Preventing Cancer by Inhibiting Fatty Acid Binding Proteins
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